JOURNAL OF PATHOLOGY, VOL. 180: 352 (1996)

LETTER TO THE EDITOR

ANTIGEN RETRIEVAL FOR IMMUNOCYTOCHEMICAL DEMONSTRATION OF ALZHEIMER PRECURSOR

PROTEIN A4 IN CNS TISSUE The publication entitled ‘Microwave antigen retrieval

for immunocytochemistry on formalin-fixed, paraffin- embedded post-mortem CNS tissue’ by McQuaid et al. ’ is a very useful contribution to this area of study. In our department we use immunocytochemistry to demon- strate many of the antigens discussed; in particular, we have had improved staining by microwave heating in citrate buffer when using anti-Alzheimer precursor protein A4 (Boehringer),’ but only when this step is followed by formic acid treatment.2 Although this combined antigen retrieval technique gives results which are an improvement on immunostaining when using formic acid pretreatment alone, we have developed a new protocol which demonstrates Alzheimer precursor protein A4 more clearly and with a greater dilution of primary antiserum. The protocol can be summarized: 1. 2.

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Dewax sections to water. Place into saturated lead thiocyanate and micro- wave to maintain a temperature of 100°C for a 10-min period.’ Stand in solution at room temperature to cool for 15 min. Rinse well in distilled water. Place in 90 per cent formic acid at room temperature for 5 min.2 Rinse well in distilled water followed by PBS. Block for endogenous peroxidase in 3 per cent H,O, in methanol.’ Rinse well in distilled water followed by PBS. Proceed with routine immunostaining using an avidin-biotin-peroxidase protocol.

By incorporating the microwave/lead thiocyanate step in our new protocol, we have improved the sensitivity of the technique without compromising specificity. In addition, the primary antiserum can now be used at a dilution of 1:200 (overnight at 4°C) where previously 1:20 was used for immunostaining with formic acid pretreatment alone and 1:50 when microwave treatment in citrate buffer was followed by formic acid treatment. Unfortunately, our modified technique did not allow improved detection of the antigen in material stored in formalin for between 5 and 20 years. We believe this information is a useful addition to the contribution of McQuaid et al.’

R. D. JOHNSEN Department of Neuropathology

Royal Perth Hospital Wellington Street, Perth

Western Australia 600, Australia

REFERENCES

1. McQuaid S, McConnell R, McMahon J, Herron B. Microwave antigen retrieval for immunocytochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue. J Pathol 1995; 1 7 6 2077216. Kitamoto T, Ogomori K, Tateishi J, Prusiner SB. Methods in laboratory investigation. Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 1987; 57: 230-236. Shi S-R, Key M, Kalra KL. Antigen retrieval in formalin-fixed, paraffin- embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytorhem 1991; 3 9 741-748.

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0 1996 by John Wiley & Sons, Ltd.